Abstract

Biotransformation and detoxification process in living organisms consists of two phases,
phase I and phase 11. Phase I involves in the introduction of functional group into
molecule while the phase I1 involves the conjugation of phase I metabolites. In phase 11,
glutathione S-transferases (GSTs; EC 2.5.1.18) has aroused much interest because of its
involvement in the biotransformation and detoxification of wide spectrum of xenobiotics
which can be from pesticides, herbicides and insecticides. The present study was
undertaken to puriG and characterized cytosolic GSTs from livers of Kedah-Kelantan
cattle (Bos indicus) and Malaysia water buffalo (Bubalus bubalis). The glutathione Stransferases
were isolated from two important livestock livers, Kedah-Kelantan cattle
(Bos indicus) and Malaysian water buffalo (Bubalus bubalis) by glutathione affinity
chromatography. The affinity-glutathione chromatography successfully purifies the
GSTs isoenzymes with 14.73% yield (62.77 purification fold) and 19.71 % yield (20.44
purification fold) for KK cattle and water buffalo livers respectively. Initial methods of
purification included centrifugation and ultracentrifugation. The affinity elution with
highest activity towards CDNB was estimated for the pI values using isoelectric
focusing method via LKB-8 100 ampholyte type (LKB Bromna) apparatus. pI values for
affinity purified KK cattle liver are 5.7 (C-34), 6.9 (C-38) and 8.8 (C-42). While for the
water buffalo liver, the pI values for glutathione affinity purified isoenzymes are 6.85
(B-23) and 7.2 (B-24). The isoenzymes were then tested using SDS-PAGE method for
purity and also to estimate the molecular weight estimation. It has been estimated that
molecular weight for water buffalo isoenzymes of B-23 was 29.3 * 0.05 kDa and B-24
was 30.74 * 0.16 kDa. The KK cattle liver isoenzymes molecular weight was estimated
with C-34 was 29.9 * 0.14; C-38 was 28.3 * 0.09 and 27.7 *0.03 for C-42. The study
showed that KK cattle liver GSTs exist as isoenzymes (PI 8.8, 6.9 and 5.7), and have
high activity towards CDNB, low towards DCNB and no activity towards the ethacrynic
acid for the substrate specificities. On the other hand, the water buffalo liver GSTs exist
as isoenzymes with pI 6.85 and 7.2. For the substrate specificities, the isoenzymes also
have high activity for CDNB, but low for DCNB and could not be detected for the
ethacrynic acid.